Metabolic Brain Disease

, Volume 31, Issue 6, pp 1391–1403 | Cite as

Delayed bradykinin postconditioning modulates intrinsic neuroprotective enzyme expression in the rat CA1 region after cerebral ischemia: a proteomic study

  • Miroslava NemethovaEmail author
  • Ivan Talian
  • Viera Danielisova
  • Sona Tkacikova
  • Petra Bonova
  • Peter Bober
  • Milina Matiasova
  • Jan Sabo
  • Jozef Burda
Original Article


Pyramidal cells in the CA1 brain region exhibit an ischemic tolerance after delayed postconditioning; therefore, this approach seems to be a promising neuroprotective procedure in cerebral postischemic injury improvement. However, little is known about the effect of postconditioning on protein expression patterns in the brain, especially in the affected hippocampal neurons after global cerebral ischemia. This study is focused on the examination of the ischemia-vulnerable CA1 neuronal layer and on the acquisition of protection from delayed neuronal death after ischemia. Ischemic-reperfusion injury was induced in Wistar rats and bradykinin was applied 2 days after the ischemic insult in an attempt to overcome delayed cell death. Analysis of complex peptide CA1 samples was performed by automated two dimensional liquid chromatography (2D-LC) fractionation coupled to tandem matrix assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) mass spectrometry instrumentation. We devoted our attention to differences in protein expression mapping in ischemic injured CA1 neurons in comparison with equally affected neurons, but with bradykinin application. Proteomic analysis identified several proteins occurring only after postconditioning and control, which could have a potentially neuroprotective influence on ischemic injured neurons. Among them, the prominent position occupies a regulator of glutamate level aspartate transaminase AATC, a scavenger of glutamate in brain neuroprotection after ischemia-reperfusion. We identified this enzyme in controls and after postconditioning, but AATC presence was not detected in the ischemic injured CA1 region. This finding was confirmed by two-dimensional differential electrophoresis followed by MALDI-TOF/TOF MS identification. Results suggest that bradykinin as delayed postconditioning may be associated with modulation of protein expression after ischemic injury and thus this procedure can be involved in neuroprotective metabolic pathways.


Cerebral ischemia Rat Hippocampus Postconditioning MALDI Proteomics 



We are indebted to Dana Jurušová for her technical assistance. We gratefully acknowledge financial support as follows. This study is the result of implementation of the project: “New possibilities of preservation of neurons in the process of delayed neuronal death by nonspecific stressors”; supported by the European Research & Development Operational Programme Funded by the ERDF - ITMS 26220220043 and by grants from the Slovak Scientific Grant Agency VEGA 2/0012/15 and 2/0045/15.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed

Supplementary material

11011_2016_9859_MOESM1_ESM.pdf (173 kb)
ESM 1 (PDF 173 kb)


  1. Arranz AM, Gottlieb M, Perez-Cerda F, Matute C (2010) Increased expression of glutamate transporters in subcortical white matter after transient focal cerebral ischemia. Neurobiol Dis 37(1):156–165. doi: 10.1016/j.nbd.2009.09.019 CrossRefPubMedGoogle Scholar
  2. Bodsch W, Barbier A, Oehmichen M, Grosse Ophoff B, Hossmann KA (1986) Recovery of monkey brain after prolonged ischemia. II Protein synthesis and morphological alterations J Cereb Blood Flow Metab 6(1):22–33. doi: 10.1038/jcbfm.1986.4 CrossRefPubMedGoogle Scholar
  3. Bonova P, Burda J, Danielisova V, Nemethova M, Gottlieb M (2013) Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain. Neurochem Int 62(6):854–860. doi: 10.1038/jcbfm.1986.4 CrossRefPubMedGoogle Scholar
  4. Boyko M, Stepensky D, Gruenbaum BF, Gruenbaum SE, Melamed I, Ohayon S, Glazer M, Shapira Y, Zlotnik A (2012) Pharmacokinetics of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase and their blood glutamate-lowering activity in naive rats. Neurochem Res 37(10):2198–2205. doi: 10.1007/s11064-012-0843-9 CrossRefPubMedGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Burda J, Martin ME, Garcia A, Alcazar A, Fando JL, Salinas M (1994) Phosphorylation of the alpha subunit of initiation factor 2 correlates with the inhibition of translation following transient cerebral ischaemia in the rat. Biochem J 302:335–338CrossRefPubMedPubMedCentralGoogle Scholar
  7. Burda J, Matiasova M, Gottlieb M, Danielisova V, Nemethova M, Garcia L, Salinas M, Burda R (2005) Evidence for a role of second pathophysiological stress in prevention of delayed neuronal death in the hippocampal CA1 region. Neurochem Res 30(11):1397–1405. doi: 10.1007/s11064-005-8510-z CrossRefPubMedGoogle Scholar
  8. Burda J, Danielisova V, Nemethova M, Gottlieb M, Matiasova M, Domorakova I, Mechirova E, Ferikova M, Salinas M, Burda R (2006) Delayed postconditioning initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain. Cell Mol Neurobiol 26(7–8):1139–1149. doi: 10.1007/s10571-006-9036-x CrossRefGoogle Scholar
  9. Burda J, Danielisova V, Nemethova M, Gottlieb M, Kravcukova P, Domorakova I, Mechirova E, Burda R (2009) Postconditioning and anticonditioning: possibilities to interfere to evoked apoptosis. Cell Mol Neurobiol 29(6–7):821–825. doi: 10.1007/s10571-009-9363-9 CrossRefPubMedGoogle Scholar
  10. Carboni L, Piubelli C, Pozzato C, Astner H, Arban R, Righetti PG, Hamdan M, Domenici E (2006) Proteomic analysis of rat hippocampus after repeated psychosocial stress. Neuroscience 137(4):1237–1246. doi: 10.1016/j.neuroscience.2005.10.045 CrossRefPubMedGoogle Scholar
  11. Chen M, Lu TJ, Chen XJ, Zhou Y, Chen Q, Feng XY, Xu L, Duan WH, Xiong ZQ (2008) Differential roles of NMDA receptor subtypes in ischemic neuronal cell death and ischemic tolerance. Stroke 39(11):3042–3048. doi: 10.1161/strokeaha.108.521898 CrossRefPubMedGoogle Scholar
  12. Cid C, Garcia-Bonilla L, Camafeita E, Burda J, Salinas M, Alcazar A (2007) Proteomic characterization of protein phosphatase 1 complexes in ischemia-reperfusion and ischemic tolerance. Proteomics 7(17):3207–3218. doi: 10.1002/pmic.200700214 CrossRefPubMedGoogle Scholar
  13. Corti V, Sanchez-Ruiz Y, Piccoli G, Bergamaschi A, Cannistraci CV, Pattini L, Cerutti S, Bachi A, Alessio M, Malgaroli A (2008) Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins. BMC Neurosci 9:36. doi: 10.1186/1471-2202-9-36 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Danielisova V, Gottlieb M, Nemethova M, Kravcukova P, Domorakova I, Mechirova E, Burda J (2009) Bradykinin postconditioning protects pyramidal CA1 neurons against delayed neuronal death in rat hippocampus. Cell Mol Neurobiol 29(6–7):871–878. doi: 10.1007/s10571-009-9369-3 CrossRefPubMedGoogle Scholar
  15. Fountoulakis M, Tsangaris GT, Maris A, Lubec G (2005) The rat brain hippocampus proteome. J Chromatogr B Analyt Technol Biomed Life Sci 819(1):115–129. doi: 10.1016/j.jchromb.2005.01.037 CrossRefPubMedGoogle Scholar
  16. Garcia-Bonilla L, Cid C, Alcazar A, Burda J, Ayuso I, Salinas M (2007) Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance. J Neurochem 103(4):1368–1380. doi: 10.1111/j.1471-4159.2007.04844.x CrossRefPubMedGoogle Scholar
  17. Gould E (2007) How widespread is adult neurogenesis in mammals? Nat Rev Neurosci 8(6):481–488. doi: 10.1038/nrn2147 CrossRefPubMedGoogle Scholar
  18. Guo ZH, Li F, Wang WZ (2009) The mechanisms of brain ischemic insult and potential protective interventions. Neurosci Bull 25(3):139–152. doi: 10.1007/s12264-009-0104-3 CrossRefPubMedGoogle Scholar
  19. Henninger N, Feldmann, RE Jr ., Futterer CD, Schrempp C, Maurer MH, Waschke KF, Kuschinsky W, Schwab S (2007) Spatial learning induces predominant downregulation of cytosolic proteins in the rat hippocampus. Genes Brain Behav 6(2):128–140. doi: 10.1111/j.1601-183X.2006.00239.x CrossRefPubMedGoogle Scholar
  20. Hossmann KA (1993) Ischemia-mediated neuronal injury. Resuscitation 26(3):225–235CrossRefPubMedGoogle Scholar
  21. Kirchner L, Chen WQ, Afjehi-Sadat L, Viidik A, Skalicky M, Hoger H, Lubec G (2008) Hippocampal metabolic proteins are modulated in voluntary and treadmill exercise rats. Exp Neurol 212(1):145–151. doi: 10.1016/j.expneurol.2008.03.014 CrossRefPubMedGoogle Scholar
  22. Kirino T (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239(1):57–69. doi: 10.1016/0006-8993(82)90833-2 CrossRefPubMedGoogle Scholar
  23. Klein JB, Gozal D, Pierce WM, Thongboonkerd V, Scherzer JA, Sachleben LR, Guo SZ, Cai J, Gozal E (2003) Proteomic identification of a novel protein regulated in CA1 and CA3 hippocampal regions during intermittent hypoxia. Respir Physiol Neurobiol 136(2–3):91–103. doi: 10.1016/S1569-9048(03)00074-0 CrossRefPubMedGoogle Scholar
  24. Klemmer P, Meredith RM, Holmgren CD, Klychnikov OI, Stahl-Zeng J, Loos M, van der Schors RC, Wortel J, de Wit H, Spijker S, Rotaru DC, Mansvelder HD, Smit AB, Li KW (2011) Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype. J Biol Chem 286(29):25495–25504. doi: 10.1074/jbc.M110.210260 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kumaran D, Hassabis D, Spiers HJ, Vann SD, Vargha-Khadem F, Maguire EA (2007) Impaired spatial and non-spatial configural learning in patients with hippocampal pathology. Neuropsychologia 45(12):2699–2711. doi: 10.1016/j.neuropsychologia.2007.04.007 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Li G, Cai F, Yan W, Li C, Wang J (2012) A proteomic analysis of MCLR-induced neurotoxicity: implications for Alzheimer's disease. Toxicol Sci 127(2):485–495. doi: 10.1093/toxsci/kfs114 CrossRefPubMedGoogle Scholar
  27. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79(9):1431–1568PubMedGoogle Scholar
  28. Lloyd-Jones D, Adams RJ, Brown TM, et al. (2010) Heart disease and stroke statistics-2010 update: a report from the American Heart Association. Circulation 121(7):e46–e215CrossRefPubMedGoogle Scholar
  29. Martin de la Vega C, Burda J, Salinas M (2001) Ischemia-induced inhibition of the initiation factor 2 alpha phosphatase activity in the rat brain. Neuroreport 12(5):1021–1025CrossRefPubMedGoogle Scholar
  30. Martin De La Vega C, Burda J, Toledo Lobo MV, Salinas M (2002) Cerebral postischemic reperfusion-induced demethylation of the protein phosphatase 2 A catalytic subunit. J Neurosci Res 69(4):540–549. doi: 10.1002/jnr.10306 CrossRefPubMedGoogle Scholar
  31. Nakajima T, Hata R, Kondo T, Takenaka S (2015) Proteomic analysis of the hippocampus in naive and ischemic-preconditioned rat. J Neurol Sci 358(1–2):158–171. doi: 10.1016/j.jns.2015.08.1530 CrossRefPubMedGoogle Scholar
  32. Otis TS, Jahr CE (1998) Anion currents and predicted glutamate flux through a neuronal glutamate transporter. J Neurosci 18(18):7099–7110PubMedGoogle Scholar
  33. Poirrier JE, Guillonneau F, Renaut J, Sergeant K, Luxen A, Maquet P, Leprince P (2008) Proteomic changes in rat hippocampus and adrenals following short-term sleep deprivation. Proteome Sci 6:14. doi: 10.1186/1477-5956-6-14 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Pulsinelli WA, Brierley JB (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10(3):267–272. doi: 10.1161/01.STR.10.3.267 CrossRefPubMedGoogle Scholar
  35. Rink C, Gnyawali S, Peterson L, Khanna S (2011) Oxygen-inducible glutamate oxaloacetate transaminase as protective switch transforming neurotoxic glutamate to metabolic fuel during acute ischemic stroke. Antiox Redox Signal 14(10):1777–1785. doi: 10.1089/ars.2011.3930 CrossRefGoogle Scholar
  36. Schmidt-Kastner R, Paschen W, Ophoff BG, Hossmann KA (1989) A modified four-vessel occlusion model for inducing incomplete forebrain ischemia in rats. Stroke 20(7):938–946. doi: 10.1161/01.STR.20.7.938 CrossRefPubMedGoogle Scholar
  37. Szatkowski M, Attwell D (1994) Triggering and execution of neuronal death in brain ischaemia: two phases of glutamate release by different mechanisms. Trends Neurosci 17(9):359–365. doi: 10.1016/0166-2236(94)90040-X CrossRefPubMedGoogle Scholar
  38. Thilmann R, Xie Y, Kleihues P, Kiessling M (1986) Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus. Acta Neuropathol (Berl) 71(1–2):88–93CrossRefGoogle Scholar
  39. Villa RF, Ferrari F, Gorini A (2013) Functional proteomics related to energy metabolism of synaptosomes from different neuronal systems of rat hippocampus during aging. J Proteome Res 12(12):5422–5435. doi: 10.1021/pr400834g CrossRefPubMedGoogle Scholar
  40. Waldner MJ, Baethmann A, Uhl E, Lehmberg J (2012) Bradykinin-induced leukocyte- and platelet-endothelium interactions in the cerebral microcirculation. Brain Res 1448:163–169. doi: 10.1016/j.brainres.2012.02.010 CrossRefPubMedGoogle Scholar
  41. Willard SS, Koochekpour S (2013) Glutamate, glutamate receptors, and downstream signaling pathways. Int J Biol Sci 9(9):948–959. doi: 10.7150/ijbs.6426 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yang JW, Czech T, Lubec G (2004) Proteomic profiling of human hippocampus. Electrophoresis 25(7–8):1169–1174. doi: 10.1002/elps.200305809 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute of Neurobiology, SASKosiceSlovakia
  2. 2.Department of Medical and Clinical Biophysics, Faculty of MedicineP. J. Safarik UniversityKosiceSlovakia

Personalised recommendations